In clinical genetic testing of biological samples such as tissue, blood, saliva, and urine, and in gene analyses of food products and plants, methods for amplifying a nucleic acid, such as PCR (polymerase chain reaction) method and LAMP (loop-mediated isothermal amplification) method, are used to efficiently analyze the target nucleic acid. These methods are effective in detecting a small amount of target. However, after amplification, the resulting solution containing a nucleic acid needs to be collected and transferred to another operation for detection, requiring opening of the reaction container, which may lead to splashing of the sample containing a nucleic acid outside the container, and also lead to false positive results due to contamination with another reaction mixture. In addition, the detection of an amplified nucleic acid requires special devices and mature testing techniques. This is why the testing is not commonly performed in sampling sites such as clinical practice and bedside. As it stands now, the testing is only carried out in clinical laboratories and specialized research institutions.
A most commonly used method for detecting an amplified nucleic acid includes separating a solution with an amplified nucleic acid using agarose electrophoresis, followed by staining with a fluorescent intercalator and observation of a specific fluorescence (Non Patent Literature 1). The fluorescence detection, however, requires expensive compounds such as a primer or an intercalator and expensive apparatus such as excitation apparatus (e.g. UV illumination device) or gel imaging apparatus. Also, the electrophoresis of an amplified nucleic acid requires a prolonged phoresis process.
Of the methods for amplifying a nucleic acid, LAMP method is advantageous in that the reaction does not require temperature cycling and proceeds at a constant temperature, and that it provides a larger amount of amplified nucleic acid than PCR method does. In particular, known methods for detecting an amplified nucleic acid obtained by LAMP method, without using fluorescence, include: a method of detection of white turbidity which is based on the formation of an insoluble magnesium salt of pyrophosphoric acid that is a by-product of the reaction; and a method of detection of changes in magnesium concentration of the reaction mixture in the presence of a metallofluorescent indicator (Patent Literature 1, Non Patent Literature 2). In fact, these detection methods, however, still require devices such as a turbidimeter or a UV illumination device because it is impossible to visually identify the amplification with absolute accuracy.
Several attempts have been made to solve the disadvantages described above. For example, a device capable of carrying out amplification and detection of a nucleic acid in a single container has been reported (Patent Literature 2). This device, however, uses an expensive labeled primer and requires complex procedures including separately adding a detecting reagent. Also reported is a device in which a dried nucleic acid amplification reagent is preliminarily added to a container to enable simple operations as well as amplification of a nucleic acid in a closed system (Patent Literature 3). However, since the reaction container needs to be opened for detection, the operations through the completion of the detection are unable to be performed in the same closed system.